Hypoxia represents a critical element in the pathogenesis of many human diseases, such as ischemic stroke, myocardial infarction and cancer and solid tumors. Understanding the mechanisms regulating hypoxia tolerance or susceptibility is essential for developing effective strategies for medical interventions. In this regard, we have generated a hypoxia-tolerant Drosophila melanogaster strain through laboratory-directed evolution (over >300 generations in descending environmental O2 levels with every several generations), sequenced their genomes and analyzed them in these flies. In parallel, we took advantage of the natural experimental evolution of humans in high-altitude regions of the world by sequencing and analyzing the whole genomes of Ethiopian and Andean highlanders. Through a comparative genomic approach combining our results with those of others, we obtained a group of evolutionarily conserved genes (28 human/23 Drosophila genes) that are potentially involved in regulating hypoxia tolerance in human highlanders and hypoxia-tolerant flies. Indeed, we discovered that ubiquitous knock-down of the expression of genes (Dm/Human), i.e., grn/GATA3, Mkk4/MAP2K4, pyd/TJP1, and shep/RBMS3, dramatically enhanced hypoxia tolerance in vivo in Drosophila. These mechanisms have a strong potential to be translated into novel targets for developing therapeutic strategies to treat hypoxia-related diseases. Our central hypothesis is that the group of evolutionary conserved genes regulates hypoxia tolerance in neurons and glial cells in humans. Our specific aims are: 1) To determine the role of evolutionarily conserved candidate genes (obtained from our Results in D. melanogaster and human highlanders) in hypoxia tolerance in vivo in whole organisms. We will determine the role of 23 candidate genes individually in hypoxia tolerance with ubiquitous or tissue/cell-specific knocking-down or overexpression using UAS/Gal4 system in D. melanogaster, and 2) To elucidate the role of evolutionarily conserved candidate genes in hypoxia tolerance in human iPSC-derived neuronal and glial cells. We will delineate the specific role of the candidate genes in protecting human neuronal and glial cells against hypoxia-induced injury by altering their expression using CRISPR/Cas9 system. We believe that this high-risk high-reward project will provide novel information about the mechanisms underlying hypoxia tolerance or vulnerability.